The goals of this project are to elucidate the mechanisms by which cells synthesize protein, and to elucidate the manner by which various genetic factors regulate such a process. For this purpose, temperature-sensitive mutants of Saccharomyces cerevisiae with altered components in the protein biosynthetic pathway are being analyzed. Two of the mutants under investigation appear to have an altered component required for chain initiation. Preliminary evidence suggests that in one of these, ts 187, the altered component is initiation factor eIF-2 which catalyzes the binding of the initiator Met-tRNAf to 40S ribosomal subunits. The eIF-2 from wild-type and temperature-sensitive cells will be purified, and their quantitative interaction with other translationa components such as Met-tRNAf, GTP, and 40S subunits will be examined. The genetic lesion in the other mutant, ts 7-45, has not been deferred exactly, within the chain initiation series of reactions; this will be carried out using the strategies and procedures developed in this laboratory, to determine whether the altered component(s) is Met-tRNA synthetase, eIF-2, 40S subunits, mRNA binding factors, 60S subunits, etc.